The present invention is directed to liposomes. More particularly, the present invention is directed to the covalent and non-covalent coupling of liposomes to proteins for purposes of in vivo targeting, or for use in diagnostics.
Liposomes are completely closed structures composed of lipid bilayer membranes containing an encapsulated aqueous volume. Liposomes may contain many concentric lipid bilayers separated by aqueous phase (multilamellar vesicles or MLVs), or may be composed of a single membrane bilayer (unilamellar vesicles).
Liposome preparation has typically been achieved by the process of Bangham et.al., (1965 J. Mol. Biol., 13: 238-252) whereby lipids suspended in organic solvent are evaporated under reduced pressure to a dry film in a reaction vessel. An appropriate amount of aqueous phase is then added to the vessel and the mixture agitated, then allowed to stand, essentially undisturbed for a time sufficient for the multilamellar vesicles to form. The aqueous phase entrapped within the liposomes may comprise bioactive agents including, but not limited to, drugs, hormones, proteins, dyes, vitamins, or imaging agents.
The current state of the art is such that liposomes may be reproducibly prepared using a number of techniques. Liposomes resulting from some of these techniques are small unilamellar vesicles (SUVs) Papahadjopoulos and Miller, Biochem. Biophys. Acta, 135, p. 624-638 (1967), reverse-phase evaporation vesicles (REV) U.S. Pat. No. 4,235,871 issued Nov. 25, 1980, stable plurilamellar vesicles (SPLV) U.S. Pat. No. 4,522,803 issued June 11, 1985, and large unilamellar vesicles produced by an extrusion technique as described in copending U.S. patent application Ser. No. 788,017, filed Oct. 16, 1985, Cullis et al., entitled "Extrusion Technique for Producing Unilamellar Vesicles", relevant portions of which are incorporated herein by reference.
One of the primary uses for liposomes is as carriers for a variety of materials, such as, drugs, cosmetics, diagnostic reagents, bioactive compounds, and the like. Such systems may be designed for both diagnostics and in vivo uses. In this regard, the ability to produce an antibody-directed vesicle would be a distinct advantage over similar undirected systems (Gregoriadis, G., Trends Pharmacol Sci, 4, p. 304-307, 1983). Useful applications would be in the selective targeting of cytotoxic compounds entrapped in vesicles to circulating tumor cells (Wolff et.al., Biochim Biophys. Acta, 802, p. 259-273 1984), or applications of these immunoglobulin-associated vesicles in the development of diagnostic assays. As is well known in the art, liposomes may be covalently coupled to proteins, antibodies and immunoglobins. Heath et.al. (Biochim. Biophys. Acta., 640, p. 66-81, 1981), describe the covalent attachment of immunoglobulins to liposomes containing glycosphingolipid. Leserman et al. (Liposome Technology, III, 1984, CRC Press, Inc., CA., p. 29-40; Nature, 288, p. 602-604, 1980) and Martin et al., (J. Biol. Chem., 257, p. 286-288, 1982) have described procedures whereby thiolated IgG or protein A is covalently attached to lipid vesicles, and thiolated antibodies and Fab' fragments are attached to liposomes, respectively. These protocols and various modifications (Martin et.al, Biochemistry, 20, p. 4229-4238, 1981; and Goundalkar et al., J. Pharm. Pharmacol. 36, p. 465-466, 1984) represent the most versatile approaches to coupling. Avidin-coupled and avidin and biotinyl-coupled phospholid liposomes containing actinomycin D have successfully targeted tumor cells expressing ganglio-N-triosylceramide (Urdal et al., J. Biol. Chem., 255, p. 10509-10516, 1980). Huang et al. (Biochim. Biophys. Acta., 716, p. 140-150, 1982) demonstrate the binding of mouse monoclonal antibody to the major histocompatibility antigen H-2 (K), or goat antibody to the major glycoprotein of Molony Leukemia Virus, to palmitic acid. These fatty acid modified IgGs were incorporated into liposomes, and the binding of these liposomes to cells expressing the proper antigens characterized. Other in vitro efforts to specific binding of liposomes coated with specific immunoglobins have been performed (Sharkey et al., Fed. Proc., 38, p. 1089, 1979). In still other coupling studies, Rahman et. al. found that tissue uptake of liposomes could be altered by attachment of glycolipids to the liposomes (J. Cell Biol., 83, p. 268a, 1979).
One aspect of the present invention is to couple biotinylated proteins such as immunoglobulins and antibodies to liposomes with covalently-attached streptavidin. Methods for this coupling are herein provided. The nature of this covalent attachment between streptavidin and the liposomes is a chemical bonding between the streptavidin, and derivatized phosphatidylethanolamine incorporated in the liposome bilayer. In a second aspect of the invention, Applicants provide a two-step method for the non-covalent coupling of these biotinylated proteins to biotinylated-phosphatidylethanolamine (PE)-containing liposomes through the same streptavidin linker. This non-covalent attachment of streptavidin and liposomes occurs through a specific association between four specific biotin binding sites on streptavidin, and the biotin. These antibody-liposome complexes bind specifically to target cells as directed by the coupled antibody Such liposomes may be made to contain bioactive agents such as drugs.
In accordance with a primary use for liposomes, the entrapment of antineoplastic agents inside liposomal bilayers has resulted in more efficacious therapy as compared to direct administration of the drug. (Forssen et al., Cancer Res., 43, p. 546, 1983; and Gabizon et al., Cancer Res., 42, p. 4734, 1982). A problem with the encapsulation of antineoplastic drugs is in the fact that many of these drugs have been found to be rapidly released from liposomes after encapsulation. This is an especially undesirable effect, in view of the fact that toxicity of these agents can be significantly reduced through liposome encapsulation as compared to direct administration. See, for example, Forssen et al. Cancer Res. 43, 546 (1983) and Rahman et al. Cancer Res., 42, 1817 (1982). Clearly, a method whereby drug could be loaded into preformed liposomes would be advantageous. To achieve this object, the invention, in accordance with one of its aspects, provides a method for loading liposomes with ionizable antineoplastic agents wherein a transmembrane potential is created across the walls of the liposomes and the antineoplastic agent is loaded into the liposomes by means of the transmembrane potential. See also U.S. patent application Ser. No. 749,161, Bally et al. entitled "Encapsulation of Antineoplastic Agents in Liposomes", filed June 26, 1985, relevant portions of which are incorporated herein by reference.
In accordance with these needs, a liposoms composition is presented which describes the use of protein-coupled liposomes which may be stored stably for an indefinite period, in a dehydrated state, with loading of the liposomes on an "as needed" basis.